Help! my colony blots are lighting up like a christmas tree

Hiranya S. Roychowdhury hroychow at nmsu.edu
Wed Feb 16 13:19:28 EST 2000


At 05:29 PM 2/16/00 -0000, R. Jayakumar wrote:
 see ther is no chance of plasmid or any
>other foreign DNA contamination.
>    I believe it could be due to bacterial debris still sticking on to the
>colony lifts on the colony lifts.
>    What do you say?
>
>jayakumar

You are exactly right! Colony blots tend to light up indiscriminatey due to
the bacterial cell debris remaining on the filter. There will always be some
background due to this but the signal-noise ratio will increase several fold
by doing what I used to do. 
I used to get good results by washing a bunch of those filters together in a
small container (in which the filters would be able to rub against each
other constantly) using the minimum volume of buffer. What actually happens
is that the remainder of the bacterial cell debris sticking to the membrane
rubs off during this. You need to change the wash buffer frequently. Try
it... you may be surprised.
This has been my experience in cosmid genomic library screening. I used to
grow the colonies directly on the membranes... but that is another protocol...


Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

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